Abstract

We present a double-helix point spread function (DH-PSF) based three-dimensional (3D) microscope with efficient photon collection using a phase mask fabricated by gray-level lithography. The system using the phase mask more than doubles the efficiency of current liquid crystal spatial light modulator implementations. We demonstrate the phase mask DH-PSF microscope for 3D photo-activation localization microscopy (PM-DH-PALM) over an extended axial range.

(a) Setup for efficiency measurement of the fabricated DH mask. The 514 nm Ar + laser line is spatially filtered. L1 and L2 are achromatic lenses of focal length f = 100 mm. The phase mask is positioned at the back focal plane of L1 (front focal plane of L2) using an x-y-z translation stage. (b) and (c) show respectively the experimental standard and DH PSF, while (d) and (e) show the corresponding, numerically simulated, PSFs. The scale bar is 150 µm.

The transverse (x-y) images for different z positions are shown for (a) the experimental PSF obtained with the fabricated DH phase mask, (b) the simulated PSF using the measured surface profile of the DH mask, and (c) the simulated PSF using the original mask design. System parameters are NA = 1.23 and M = 137X. The scale bar is 1 µm. (d) Calibration curve showing the plot of the DH-PSF rotation angle versus the axial position of the emitter, for the experimental PSF and, for comparison, the same plot for the ideal PSF obtained from the mask design.

(a) An image of the quantum dot. The scale bar is 500 nm. The 3D position of the quantum dot was estimated using the Angle Estimator. Panels (b), (c) and (d) show the histogram of 150 position estimations along x, y and z dimensions.

3D single PA-GFP-tubulin molecule localizations in PtK1 cells using PM-DH-PALM with a surface relief phase mask. (a) 3D image of the reconstructed region. The colormap encodes depth. (b) image of one detected molecule (see text for details). The scale bar is 500 nm. The estimation of the 3D position for this single molecule was done using the Angle Estimator. (c), (d) and (e) show the histogram of 37 position estimations along x, y and z.

Average performance of the PM-DH-PALM experiment: (a) Histogram of the number of molecules observed with a given number of photons detected in the two DH lobes. Median number of photons is 911. (b)-(d) show the histograms of 3D position estimations of identified molecules with detected photons in the range 850 to 950. The observed precision was σ(X,Y,Z) = (33 nm, 30 nm, 76 nm).